ABSTRACT/PROJECT SUMMARY Although often studied as distinct entities, transcription and RNA processing are intricately linked in eukaryotes. Our previous results in budding and fission yeasts show that the spliceosome can quickly and efficiently remove introns as soon as Pol II synthesizes them. Nevertheless, sometimes splicing is completely suppressed, rendering transcripts ?dead end?. These unspliced nascent transcripts fail to undergo polyA cleavage and are degraded. We hypothesize that the balance between ?productive? (spliced and polyadenylated) and ?dead-end? transcripts determines gene expression in mammalian cells. Furthermore, this mechanism is likely crucial under conditions of stress, because transcriptional readthrough is a frequent feature of cellular stresses induced by infection, cancer, osmotic and oxidative stress, and other conditions. The application brings together the complementary expertise of two investigators who are responding to an RFA from NHLBI on normal biological mechanisms that provide cells with resilience. Dr. Neugebauer is a biochemist with expertise in transcription and splicing, while Dr. Pillai is a hematopoietic biologist with expertise in generation of erythroid populations and their characterization. Our proposal investigates the coupling between transcription and RNA processing during production of red blood cells (erythropoiesis or EP). Mature enucleated red blood cells emerge from immature hematopoietic progenitors after undergoing a highly regulated differentiation program guided by numerous exogenous signals. This differentiation is characterized by dramatic changes in the transcriptome, resulting in a mature red cell that is essentially a hemoglobin factory. b-globin, the most abundant transcript in mature erythroid cells, has served as a critical model for pioneering studies in pre-mRNA splicing and mRNA stability. We hypothesize that positive and negative feedback between splicing and transcription are important determinants of erythroid maturation, which must be resilient to physiological conditions (e.g. pregnancy, high altitude) that cause tissue hypoxia. The resulting ?Stress EP? increases red cell production in order to deliver more oxygen to the tissues. We therefore propose to investigate co-transcriptional splicing dynamics, using erythropoiesis as a model system. We will implement two custom nascent RNA-Seq strategies developed in the Neugebauer lab: Single Molecule Intron Tracking (SMIT) and long read sequencing of nascent RNA. In Aim 1, we will utilize an in vitro culture model of human erythropoietic differentiation in which primary CD34+ cells are cultured with erythropoietin and other trophic factors to generate erythroid cells and test the above hypotheses. Aim 2 will explore how co-transcriptional RNA processing may contribute to transcriptomic changes during stress EP. This study thereby pioneers experimental systems that will allow us to pinpoint gene regulatory mechanisms that rely on transcription and splicing to maintain cellular homeostasis.